Diagnostic testing Used to confirm or rule out a known suspected

(mutation) genetic disorder in a symptomatic person.

Predictive testing Tells an asymptomatic individual if he/she carries a

mutation that will cause, or put them at higher risk for, a disease later in life.

Newborn screening Detects common disorders in newborns, where

immediate treatment can prevent dangerous symptoms

Carrier testing Tells a person whether or not he carries a mutation

that could be passed on to his offspring. One can be a carrier, but not be at risk for a disease (recessive genes)

? ? ? ?

Genetic Testing – Why test DNA?

Human KaryotypeGenome: all genetic material in a nucleus.

Humans:

22 pairs of autosomes

1 pair of sex chromosomes

X & Y

DNA in the Cell

chromosome

cell nucleus

Double stranded DNA molecule

Individual nucleotidesEvery body cell has 46 chromosomes.

Every sex cell (egg/sperm) has 23 chromosomes.

Each nucleotide contains:

Nitrogenous Base

Sugar (deoxyribose)

Phosphate Group

4 different bases make up the steps of the ladder

A Adenine T Thymine G Guanine C Cytosine

Base Pairing Rules

Sugar-Phosphate backbone

G-C A-T

Each rung on the ladder is called a

1 base pair (bp)

2 base pair (bp)

3 base pair (bp)

How many base-pairs are there in the human genome? ( in 1 nucleus / 46 chromosomes)

approximately 3 billion

base-pair

A C G G T A A G C A T A G C C G T G A G T T T A C C A G

The Genetic Code A series of G’s A’s C’s & T’s

How much of your DNA is identical to everyone else?

99.4%

DNA

RNA

Protein

Function

• Some genes are expressed in every cell• …But the proteins they code for may have different

effects/interactions on each cell type. • Some genes are expressed only in a particular type of

cell, giving that cell type its unique structure and function

DNA

Protein

ProteinFunction

Levels of Genetic Testing

normal mutated

Analysis of whole chromosomes – for large changes; extra chromosome, very large deletions or insertions

atcgatcgatcg atcgaAcgatcgAnalysis of sequence – for small changes; mutations in the sequence, small deletions or insertions

Analysis of protein shape – for any change that may affect the folding of the protein

Analysis of protein function – if the functional protein is supposed to make something, then some tests can detect the presence or absence of the product

X X

Organization of the Human Genome

GeneGene – region that – region that encodes for a encodes for a protein or a traitprotein or a trait

Eye colorEye color

DimplesDimples

FrecklesFreckles

Blood typeBlood type

MOM DAD

Organization of the Human GenomeEvery gene can have alternate forms

called allelesBlue eyesBlue eyes

DimplesDimples

No FrecklesNo Freckles

Type IType IAA

For every gene in your DNA you have… 2 alleles.

Homozygous (same)

Heterozygous (different) MOM DAD

Brown eyesBrown eyes

No dimplesNo dimples

FrecklesFreckles

Type IType IBB

aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Normal

Examples of Mutations in the DNA Sequence…

aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgataatcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Single base pair mutation

Examples of Mutations in the DNA Sequence…

(Sickle cell anemia)

aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatggtcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatggggatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacatttttttttaaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Multiple mutations

Examples of Mutations in the DNA Sequence…

(Diabetes, susceptibility to breast cancer)

aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Deletion

Examples of Mutations in the DNA Sequence…Examples of Mutations in the DNA Sequence…

(Cystic fibrosis)

aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaaaaaaaaaaaaaaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Insertion

Examples of Mutations in the DNA Sequence…

(Huntington’s disease)

STR’s Short Tandem Repeats (smaller segments with fewer repeats)

CGAA CGAA CGAA CGAA CGAA CGAA CGAA CGAA CGAA CGAA CGAA

VNTR’s Variable Number Tandom Repeats (larger segments with many repeats)

GGACTAATATCTATTCCCTAATATGACTAA

AATATTTCGGACTAGATATCTTTCGGACTTA

TCTATTCGGGAGCCGCTACCCGTG…

…X 1000

A gene that includes a tandem repeat sequence can have many different alleles in a population. Ex:

Allele 1: (6 repeats)

GAACT ATTA ATTA ATTA ATTA ATTA ATTA CGTGCAGGCT

Allele 2: (5 repeats)

GAACT ATTA ATTA ATTA ATTA ATTA CGTGCAGGCT

Allele 3: (7 repeats)

GAACT ATTA ATTA ATTA ATTA ATTA ATTA ATTA GTGCAGGCT

How do we “look” for a mutation in a DNA sequence?

• Sequencing

• Agarose Gel Electrophoresis

• PCR

• RFLP analysis

• Probes– Dot blot– Microarray

PCR Procedure1. Heat DNA strands, causing strands to unzip.

2. Cool, add primer pairs, short sequences of DNA (starter pieces) that will bind to the complementary sequences at the beginning of the separated DNA strands. Annealing

3. Add: A. DNA polymerase - “replicator machine”

B. Nucleotides (AGCT) building blocks for new DNA

C. Heat to around 75° C for the completion.

Make copies (extend primers)

Starting DNA Template

5’

5’

3’

3’

5’

5’

3’

3’

Add primers (anneal) 5’3’

3’5’

Forward primer

Reverse primer

DNA Amplification with the Polymerase Chain Reaction (PCR)

Separate strands

(denature)

5’

5’3’

3’

In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created

In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created

PCR Copies DNA Exponentially through Multiple Thermal Cycles

Original DNA target region

Thermal cycleThermal cycleThermal cycle

Each cycle takes less than 2 minutes from start to finish.

PCR Electrophoresis

Electrophoresis

At least one lane must contain a DNA ladder (a set of molecules with known sizes.

Each band in these lanes contains a known length of DNA.

These ladders are used to determine the length of the DNA in fragments in other lanes.

From the size of the fragment, the number of repeat sequences can be determined.

• Each person has 2 alleles for a gene. M/D

• The resulting pattern of an STR sequence for a single gene has either 1 or 2 bands

• Can be homozygous or heterozygous for each gene.

• 1 band = homozygous – both fragments same length

• 2 bands = heterozygous – different fragment lengths

17 repeats

16 repeats

15 repeats

14 repeats

13 repeats

12 repeats

11 repeats

10 repeats

9 repeats

8 repeats

7 repeats

6 repeats

Available Predictive Tests

Cystic Fibrosis

Tay Sachs Disease

Lou Gherig’s Disease (ALS)

Huntington’s Disease

Catastrophically high cholesterol

Rare Cancers

Inherited susceptibilty to cancerBreast Cancer Colon Cancer Thyroid Cancer